In this renewal we take full advantage of our expertise in molecular and functional analyses of splice variants of the voltage-gated N-type Ca channel alph-1 subunit. We outline an integrated approach to study the molecular basis of functional diversity among N and L-type Ca channels expressed with regional variation in the nervous system. At center stage is the role of alternative splicing in generating this diversity. By systematically analyzing RNA isolated from distinct regions of the nervous system and combining this information with analysis of newly released human Ca channel alpha 1B sequences, we discern the extent of alternative splicing among N-type Ca channel alpha2.2-1B and L-type Ca channel alpha1.3-1D subunit gene families (aims 1 and 3). The functional impact of alternative splicing on the biophysical properties of N-type and L-type Ca channels will be elucidated. Expression systems have already been developed in our lab for this purpose. Where new techniques are proposed such as in situ hybridization, we have established key collaborations with recognized experts. In aim 2 we focus on beta-subunit-specific modulation of associated alha-1 subunits. With the use of brief, high frequency stimulation including action potential wave-forms we will investigate frequency-dependent inactivation of N channels. These more physiologically relevant stimuli will allow us to better predict how different alpha1/beta-subunit combinations might impact voltage-dependent Ca entry at the synapse. There are several potential benefits of our studies. (i) A comprehensive view of alternative splicing in N and L-type alpha 1 genes will significantly advance our understanding of the molecular origins of functional diversity in Ca signaling. (ii) Alternatively spliced exons that are under cellular control are invariably located in protein domains important for regulating function. By studying these domains we gain unique insights into structure/function relationships in Ca channel alpha 1 subunits and, more generally, in other voltage-gated ion channels that share the canonical six transmembrane domain structure. (iii) L-type and N-type Ca channels are important therapeutic targets. Alternative splicing has the potential to create pharmacological diversity among members of a single alpha 1 gene. Consequently, the identification of tissue-specific sites of alternative splicing may offer new strategies for improving specificity of drug action.